.

SURFACE ENTRAPMENT OF CHITOSAN ON 3D PRINTED POLYLACTIC

ACID SCAFFOLD

AUGUST 2018

A thesis submitted in fulfilment of the

requirements for the award of the degree of

Master of Philosophy

NOR HIDAYAH BINTI ZAKARIA

School of Chemical and Energy Engineering

Faculty of Engineering

Universiti Teknologi Malaysia

iii

To the persons that always there during those difficult and trying times,

Zakaria Hj Mohd Saad, Radiah binti Mohd Som, Nadirul Hasraf Mat Nayan and

Nadhif Hasraf Nadirul Hasraf

DEDICATION

iv

In the name of Allah S.W.T, the most gracious and the most merciful.

Alhamdulillah, with the utmost blessing from Allah and in the remembrance to our

prophet Muhammad P.B.U.H His most beloved messenger of all time, the path of

my master degree has come to a completion.

I would like to take this opportunity to show my deepest gratitude to Dr.

Saiful Izwan bin Dato’ Abd Razak who had stand in front of me to led my may in

achieving my master degree. This thesis has become a reality due to his wholly

support and knowledge, guidance and moral support a along with the will from

Allah S.W.T. The appreciation also goes to my co-supervisor, Assoc. Prof. Dr.

Abdul Razak Rahmat for his provision on supervising my research.

Not to be forgotten my beloved parents, Zakaria Bin Mohd Saad and Radiah

Binti Mohmad Som, my lovely husband and son, Nadirul Hasraf Bin Mat Nayan and

Nadhif Hasraf Bin Nadirul Hasraf for placing their highest belief in me to complete

this thesis. Same thanks to my family members and friends who are willing to bear

with me through thick and torn together in making my dream come true. Finally,

tremendous assistance from all of my colleagues in Polymeric biomaterials lab would

not be forgotten and always be in my thought and prayers.

ACKNOWLEDGEMENT

v

This thesis reports a surface entrapment of chitosan on 3D printed PLA scaffold

which has the potential use in promoting bone regeneration. The 3D scaffold was

designed using SolidWorks and printed by Up Plus 3D printer and then incorporated

with chitosan. The entrapped scaffold time was varied from 5 to 90 s. The scaffold was

characterized in respect of its mechanical and surface properties. Compressive test

showed a higher compressive modulus properties in neat 3D printed PLA scaffolds

and an optimum value of 22248 MPa at 15 s of chitosan immersion. The Fourier-

transform infrared spectroscopy peak revealed an existence of biomacromolecule and

new absorption peaks at 3357 and 1618 cm-1 compared to neat PLA on the scaffold

while water contact angle showed an increase in hydrophilicity as entrapment time

increased. The confocal laser scanning microscopy revealed the existence of

entrapment areas approximately 8𝜇m in depth. The scanning electron microscopy

showed clearly 3D scaffold with high porosity, uniform distribution chitosan and a

controlled and repetitive architecture on entrapped 3D printed scaffold. Immersion of

neat and entrapped 3D printed PLA scaffold in simulated body fluid for 14 days

resulted the formation of fully covered apatite layers on the surface of entrapped PLA

scaffold whereas no change was observed in neat PLA scaffold. Overall, the

mechanical and surface properties results showed the suitability of the combination of

method and materials to develop 3D porous scaffold and their initial biocompatibility,

both being valuable characteristic for tissue engineering applications.

ABSTRACT

vi

Tesis ini melaporkan tindakbalas pemerangkapan permukaan kitosan pada

perancah PLA bercetak tiga dimensi (3D) yang mempunyai keupayaan untuk

digunakan bagi menggalakkan pertumbuhan semula tulang. Perancah 3D direka

menggunakan SolidWorks dan dicetak oleh pencetak 3D Up Plus yang

mengandungi kitosan. Masa pemerangkapan adalah dari 5 saat ke 90 saat. Perancah

dicirikan terhadap sifat mekanik dan sifat permukaannya. Ujian mampatan

menunjukkan sifat modulus mampatan yang lebih tinggi dalam perancah PLA

dicetak 3D dengan nilai optima 2248 MPa pada 15 s rendaman kitosan. Puncak

spektroskopi inframerah transformasi Fourier menunjukkan kewujudan puncak

biomakromolekul dan puncak penyerapan baharu pada 3357 dan 1618 cm-1

berbanding dengan PLA tanpa pemerangkapan manakala sudut sentuhan air

menunjukkan peningkatan hidrofilik bila meningkatnya masa pemerangkapan.

Mikroskop imbasan laser konfokal menunjukkan kewujudan kawasan

pemerangkapan sedalam kira-kira 8 μm. Mikroskop elektron imbasan bagi

perancah 3D yang memerangkap kitosan jelas menunjukkan permukaan berliang

yang saling berkait, penyebaran kitosan yang seragam dan seni bina yang terkawal

dan berulang. Hasil rendaman kedua - dua perancah di dalam cecair badan simulasi

selama 14 hari menghasilkan pembentukan lapisan mineral di permukaan perancah

PLA yang melalui proses pemerangkapan manakala tiada pembentukan yang dapat

dilihat berlaku pada perancah 3D biasa. Secara keseluruhan, hasil keputusan ujikaji

sifat mekanik dan sifat permukaan menunjukkan kesesuaian di antara kombinasi

kaedah dan bahan di dalam pembentukan perancah berpori 3D dan biokeserasian

awal, di mana kedua-duanya menjadi ciri-ciri penting untuk aplikasi kejuruteraan

tisu.

ABSTRAK

vii

TABLE OF CONTENTS

CHAPTER

TITLE PAGE

DECLARATION ii

DEDICATION iii

ACKNOWLEDGEMENT iv

ABSTRACT v

ABSTRAK vi

TABLE OF CONTENTS vii

LIST OF TABLES viii

LIST OF FIGURES ix

LIST OF ABBREVATIONS x

1 INTRODUCTION 1

1.1 Overview 1

1.2 Problem Statement 3

1.3 Objectives of the Study 4

1.4 Scope of Study 5

2 LITERATURE REVIEW 6

2.1 Tissue Engineering 6

2.2 Scaffold in Tissue Engineering 7

2.2.1 Scaffold Development 8

2.2.2 Characteristics of Scaffolds 8

viii

2.2.3 Scaffold Material 10

2.3 Polylactic Acid (PLA) 11

2.4 3D Printing in Biomedical Application 13

2.4.1 3D Printing Technique 13

2.5 Chitosan 14

2.5.1 Surface Modification by Entrapment of Chitosan 16

2.5.2 Influence of Chitosan on Growth of HA 19

2.6 Summary of Literature Review 21

3 MATERIALS AND METHODOLOGY 22

3.1 Material and Equipment 22

3.1.1 Material and reagents 22

3.1.2 3D Printer 23

3.2 Experimental Methods 24

3.2.1 Designing and Drawing od scaffold 24

3.2.2 Scaffold Fabrication 25

3.2.3 Surface Modification Process 26

3.3 Characterization and Testing 27

3.3.1 Mechanical Testing 27

3.3.2 Scanning Electron Microscopy 28

3.3.3 Water Contact Angle 28

3.3.4 Confocal Laser Scanning Microscopy (CLSM) 28

3.3.5 Fourier Transform Infrared Spectroscopy (FTIR) 29

3.3.6 In-vitro Biomineralization Test 29

3.3.7 Flow diagram of the research methodology 30

ix

4 RESULTS AND DISCUSSIONS 31

4.1 Compressive Test of 3D Printed PLA/chi Scaffold 31

4.2 Water Contact Angles of PLA/chi scaffold 32

4.3 Morphological of 3D Printed PLA/chi Scaffold 34

4.4 FTIR of PLA/chi Scaffold 35

4.5 Confocal Laser Scanning Microscopy (CLSM) of PLA/chi

Scaffold 36

4.6 In-Vitro Biomineralization 39

5 CONCLUSION 43

5.1 Conclusion 43

5.2 Future Work 44

REFERENCES 45

Appendix A 54

x

LIST OF TABLES

TABLE NO.

TITLE PAGE

2.1 The Characteristics of an Effective Scaffold 10

3.1 Chemicals and Reagents 24

3.2 Specification of 3D printer 25

xi

LIST OF FIGURES

FIGURE NO.

TITLE PAGE

2.1 The approach of using cell-scaffold conduct (Gualandi,

2011)

8

2.2 Repeating unit of PLA (Gruber et al., 2003) 13

2.3 The structure of chitin and chitosan 16

2.4 Schematic of surface properties of the biomaterial

implanted in the host (Shen, 2007)

18

2.5 Schematic of entrapment process in PLA film (H.Zhu et

al., 2002)

20

3.1 Scaffold design by SolidWork 26

3.2 3D printing and surface modification process 28

3.3 Flow diagram of the research methodology 31

4.1 Compressive modulus of neat PLA scaffold and

entrapped PLA scaffold

34

4.2 WCA of (a) neat PLA (b) PLA/chi5 (c) PLA/chi10 (d)

PLA/chi15 (e) PLA/chi30 (f) PLA/chi60 (g) PLA/chi90

36

4.3 SEM images of (a) neat PLA scaffold (b) PLA/chi15

scaffold

37

4.4 FTIR spectra of (a) neat PLA (b) PLA/chi15 38

4.5 CLSM images of (a) top image (b) single printed layer

of PLA/chi15 scaffold

40

4.6 Fluorescence intensity on the edge of the PLA/chi15

scaffold

40

4.7 SEM images of (a,b) neat PLA soaked for 14 days (c,d)

PLA/chi15 soaked for 1 day (e,f) PLA/chi15 soaked for

7 days (g-i) PLA/chi15 soaked for 14 days

43

xii

4.8 Proposed Illustration of HA formation on 3D printed

scaffold

44

xiii

LIST OF ABBREVATIONS AND SYMBOLS

PLA - Poly (lactic acid)

SEM - Scanning Electron Microscopy

3D - Three Dimensional

AM - Additive manufacturing

CAD - Computer Aided Design

FDA - Food and Drug Administration

HA - Hydroxyl apatite

SBF - Simulated Body Fluid

RP - Rapid Prototyping

OH - Hydroxide Group

NaOH - Sodium Hydroxide

stl - sterealitography

CLSM - Confocal Laser Scanning Microscopy

SPIN - Surface Interpreting Network

ECM - Extracellular matrix

Ca/P - Calcium/Phosphate

FTIR - Fourier Transform Infrared Spectroscopy

WCA - Water contact angle

INTRODUCTION

Overview

Rapid prototyping (RP), is an attractive tool in fabrication of scaffold in tissue

engineering (Shirazi et al., 2015). This technique can produce a complicated design

with well-defined structures and reproducible architectures (Gross et al. 2014). It has

open the possibility in making scaffold considering biomedical diagnostic from

individual patient and needs (Ventola, 2014).

Modern 3D printing with the aid of computer design and automatic printing

technology can tailor made the fabrication of scaffold (Guvendiren et al., 2016). Most

other techniques fails to produce this desired properties due to lack the capability of

computer design. This includes the effects of geometry/architecture on cell response,

and for computer modeling of the scaffold’s behavior (Sears et al., 2016). By using

3D printer, an improved mechanical performance of three-dimensional (3D) structures

also can be obtained (Serra et al., 2013).

2

Polylactic acid (PLA) are one of the popular biomaterial reported to be used

in 3D printing specified in Fused Deposition Modeling (FDM) technologies due to its

low cost, nontoxicity and ease of processability (Guvendiren et al., 2016) . Though

there are few reports on the use of PLA biomaterial for production of 3D printed

scaffolds, serious concerns are on PLA long-term biocompitability due to production

of acidic by product, its hydrophilicity (Dong et al., 2010) and lack of functional group

for covalent cell-recognition signal molecules in the PLA to promote cell adhesion

(Zhu, 2002). Those drawbacks can lead to tissue inflammation and cell death. To

remedy this, PLA often combined with other bioactive fillers such as calcium

phosphate glass (Serra et al., 2013), 45S5 bioactive glass (Estrada et al., 2017),

nanocellulose (Wang et al., 2017) and hydroapatite (Corcione et al., 2017). However,

since both material have a distinct physical, chemical and biological properties

(Barbosa et al., 2010), and the 3D fabrications used are costly since it requires

preliminary processing compatibility with the 3D printer setup.

Other method to combat this drawback are by modifying PLA scaffold using

surface modification technique either by physio sorption, covalent bonding (Serra et

al., 2013) or by creating surface interpenetrating networks (Quirk et al., 2001).

Chitosan are one of the biomacromolecules that have been successfully modifies the

surface of PLA (Cui et al., 2003). Modifying PLA with chitosan can improve its

osteoconductivity, biocompatibility and its suitable degradation rate (Dutta et al.,

2004). To date, this method still lack of reports especially on the mechanical integrity

and surface properties of the modified scaffold.

With the aid of this feasible method, degradable porous material scaffold were

designed to integrate the cell proliferate and tissue regeneration. This is one way to

deal with repair and recovery of cell and tissue by establish upon the utilization of

polymer scaffold which serve to bolster, strengthen and at times sort out the removing

tissue (Madihally, 1999). The mechanical expect for scaffold is also important as a

mass transport biological delivery and tissue regeneration (Hollister, 2005).

3

Researchers had studied the experimental and clinical studies of 3D printed

scaffold for biomimetic application in scaffold bioresorbable resource as well as its

design. This work describe the fabrication of PLA-based scaffold by 3D printer.

Chitosan molecules were entrapped in PLA to obtain 3D scaffold with high mechanical

and high bioactive properties. The structures obtained are characterized in terms of

mechanical behavior, surface properties as well as in vitro biomineralization studies.

Problem Statement

PLA is widely used in biomedical application and as biodegradable polymer

that been approved by FDA (Almeida, 2013). Most studies focus on PLA mechanical

and morphological improvement. Even PLA have been extensively studied especially

in scaffold fabrication, the fabrication of PLA scaffold specifically through nozzle-

based system are scarcely reported. Plus the resulting printed PLA scaffold usually

produced lack in biological moieties which require additional process to activate the

biological sites (Zhu et al., 2002). Thus making the study of the production of PLA

scaffold via commercial 3D were limited to some extent.

PLA is problematic for tissue and implant engineering application due to its

absence of biologically active site. Production of a stable and biocompatible PLA

scaffold are limited since it may modifies main polymer structure or may require a

subsequent process solvent removal from its final structure (Serra et al., 2013).

According to Zhu (2002), one of the strategies to render this properties is to design

back the polymer backbone that have function monomer unit by introducing

functional group on the surface or the polymer backbones.

It is reported, the most straight forward method is coating the surface of the

biomaterial with bioactive molecules (Li et al., 2009). This method however it is

problematic due to it instability of the layers (coat) thus restrict it further application

4

in biomedical field (Shen, 2007). Since the PLA printed scaffold are limited in its

biocompatibility, this limitation can be overcome with incorporation of chitosan (Cui

et al., 2003). Chitosan are one of bioactive molecules that have been proved to have

great osteoconductivity, biocompatibility, suitable degradation rate and minimal

foreign body reaction (Cui et al., 2003; Dutta et al., 2004; Collection et al., 2000; Shen

et al., 2000). Incorporation of chitosan in PLA structure will lead a more biocompatible

scaffold structure (Rogina et al., 2016).

Porous structure of 3D printed can promote faster healing. Though the printed

scaffold method can produce a uniform and repetitive porosity, various cumbersome

factors should be taken into consideration to design a porous and stable structure such

as pore size and the exposition of elevated temperature of the polymer which may lead

to denaturation and toxic production of PLA scaffold (Pfister et al., 2003). Therefore,

it is important have suitable intrinsic material properties but also geometry of the 3D

scaffolds to design the new surface and tailor macrophage activation toward

regenerative pathway (Zhu, 2002).

Being relatively new in the tissue engineering field, 3D printed PLA scaffold

with incorporation of biomacromolecules has many unexplored features and

characteristics. By combining 3D printing method together with suitable

biodegradable polymers, fabrication of 3D scaffold it is possible with well-

distinguished geometric, different characteristics and allowing the study of the effect

of surface entrapment to those cell responses.

Objectives of the Study

There are three objectives to be achieved in this study. There are as following:

a) To design and prepare 3D printed PLA scaffold with surface entrapment of

chitosan.

5

b) To study the effects of chitosan entrapment on the compressive strength

and surface properties of the PLA modified scaffold

c) To investigate the preliminary in vitro biomineralization of the scaffold.

Scope of Study

In order to satisfy all the outlined objectives, the scopes of this research are

undertaken according to the following.

Initially, PLA scaffold were designed and fabricated using 3D printer. The

scaffold were first design using SolidWork drawing before converted to suitable

format for the 3D printer system. The produced scaffold are characterized for its

morphology and appearance.

Next step is to produce a bioactive scaffold by entrapment of chitosan in the

surface of the 3D printed scaffold. The scaffolds were immersed in chitosan solution

for period of 5, 10, 15, 30, 60 and 90s. The resulting entrapped scaffold were evaluated

in terms of its mechanical and morphological properties. Mechanical test conducted

were compressive strength of the scaffold. Other than that, surface properties such as

FTIR, WCA and SEM were also being evaluated.

Consequently, the scaffold were tested for in-vitro bio mineralization to test its

bioactivity. This was done through immersion in simulated body fluid solution (SBF)

and followed by evaluation of hydroxyl apatite growth on the sample.

45

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LIST OF PUBLICATIONS

1. Zakaria, N.H., Mat Nayan, N.H., Razak, S. I. A. (2017). Effect of

Biomacromolecules on the surface porosity and hydrophilicity of Polylactic Acid

Scaffold, Asia international Multidisplinary conference, AIMC-2017-STE-971.